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. 1996 Mar;178(5):1412–1419. doi: 10.1128/jb.178.5.1412-1419.1996

Genetic relationship between the 53- and 49-kilodalton forms of exoenzyme S from Pseudomonas aeruginosa.

T L Yahr 1, J T Barbieri 1, D W Frank 1
PMCID: PMC177816  PMID: 8631719

Abstract

Exoenzyme S is an ADP-ribosylating extracellular protein of Pseudomonas aeruginosa that is produced as two immunologically related forms, a 49-kDa enzymatically active form and a 53-kDa inactive form. The postulated relationship between the two proteins involves a carboxy-terminal proteolytic cleavage of the 53-kDa precursor to produce an enzymatically active 49-kDa protein. To determine the genetic relationship between the two forms of exoenzyme S, exoS (encoding the 49-kDa form) was used as a probe in Southern blot analyses of P. aeruginosa chromosomal digests. Cross-hybridizing bands were detected in chromosomal digests of a strain of P. aeruginosa in which exoS had been deleted by allelic exchange. A chromosomal bank was prepared from the exoS deletion strain, 388deltaexoS::TC, and screened with a probe internal to exoS. Thirteen clones that cross-hybridized with the exoS probe were identified. One representative clone contained the open reading frame exoT; this open reading frame encoded a protein of 457 amino acids which showed 75% amino acid identity to ExoS. The exoT open reading frame, cloned into a T7 expression system, produced a 53-kDa protein in Escherichia coli, termed Exo53, which reacted to antisera against exoenzyme S. A histidine-tagged derivative of recombinant Exo53 possessed approximately 0.2% of the ADP-ribosyltransferase activity of recombinant ExoS. Inactivation of exoT in an allelic-replacement strain resulted in an Exo53-deficient phenotype without modifying the expression of ExoS. These studies prove that the 53- and 49-kDa forms of exoenzyme S are encoded by separate genes. In addition, this is the first report of the factor-activating-exoenzyme-S-dependent ADP-ribosyltransferase activity of the 53-kDa form of exoenzyme S.

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Selected References

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  1. Adhya S., Gottesman M. Control of transcription termination. Annu Rev Biochem. 1978;47:967–996. doi: 10.1146/annurev.bi.47.070178.004535. [DOI] [PubMed] [Google Scholar]
  2. Bjorn M. J., Pavlovskis O. R., Thompson M. R., Iglewski B. H. Production of exoenzyme S during Pseudomonas aeruginosa infections of burned mice. Infect Immun. 1979 Jun;24(3):837–842. doi: 10.1128/iai.24.3.837-842.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Coburn J. Pseudomonas aeruginosa exoenzyme S. Curr Top Microbiol Immunol. 1992;175:133–143. doi: 10.1007/978-3-642-76966-5_7. [DOI] [PubMed] [Google Scholar]
  4. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Forsberg A., Wolf-Watz H. Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression. J Bacteriol. 1990 Mar;172(3):1547–1555. doi: 10.1128/jb.172.3.1547-1555.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frank D. W., Iglewski B. H. Cloning and sequence analysis of a trans-regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa. J Bacteriol. 1991 Oct;173(20):6460–6468. doi: 10.1128/jb.173.20.6460-6468.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frank D. W., Nair G., Schweizer H. P. Construction and characterization of chromosomal insertional mutations of the Pseudomonas aeruginosa exoenzyme S trans-regulatory locus. Infect Immun. 1994 Feb;62(2):554–563. doi: 10.1128/iai.62.2.554-563.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frithz-Lindsten E., Rosqvist R., Johansson L., Forsberg A. The chaperone-like protein YerA of Yersinia pseudotuberculosis stabilizes YopE in the cytoplasm but is dispensible for targeting to the secretion loci. Mol Microbiol. 1995 May;16(4):635–647. doi: 10.1111/j.1365-2958.1995.tb02426.x. [DOI] [PubMed] [Google Scholar]
  9. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  10. Hovey A. K., Frank D. W. Analyses of the DNA-binding and transcriptional activation properties of ExsA, the transcriptional activator of the Pseudomonas aeruginosa exoenzyme S regulon. J Bacteriol. 1995 Aug;177(15):4427–4436. doi: 10.1128/jb.177.15.4427-4436.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Iglewski B. H., Sadoff J., Bjorn M. J., Maxwell E. S. Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3211–3215. doi: 10.1073/pnas.75.7.3211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Knight D. A., Finck-Barbançon V., Kulich S. M., Barbieri J. T. Functional domains of Pseudomonas aeruginosa exoenzyme S. Infect Immun. 1995 Aug;63(8):3182–3186. doi: 10.1128/iai.63.8.3182-3186.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kudoh I., Wiener-Kronish J. P., Hashimoto S., Pittet J. F., Frank D. Exoproduct secretions of Pseudomonas aeruginosa strains influence severity of alveolar epithelial injury. Am J Physiol. 1994 Nov;267(5 Pt 1):L551–L556. doi: 10.1152/ajplung.1994.267.5.L551. [DOI] [PubMed] [Google Scholar]
  14. Kulich S. M., Frank D. W., Barbieri J. T. Expression of recombinant exoenzyme S of Pseudomonas aeruginosa. Infect Immun. 1995 Jan;63(1):1–8. doi: 10.1128/iai.63.1.1-8.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kulich S. M., Frank D. W., Barbieri J. T. Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388. Infect Immun. 1993 Jan;61(1):307–313. doi: 10.1128/iai.61.1.307-313.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kulich S. M., Yahr T. L., Mende-Mueller L. M., Barbieri J. T., Frank D. W. Cloning the structural gene for the 49-kDa form of exoenzyme S (exoS) from Pseudomonas aeruginosa strain 388. J Biol Chem. 1994 Apr 8;269(14):10431–10437. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Mekalanos J. J., Swartz D. J., Pearson G. D., Harford N., Groyne F., de Wilde M. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature. 1983 Dec 8;306(5943):551–557. doi: 10.1038/306551a0. [DOI] [PubMed] [Google Scholar]
  19. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  20. Nicas T. I., Iglewski B. H. Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S. Infect Immun. 1984 Aug;45(2):470–474. doi: 10.1128/iai.45.2.470-474.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schweizer H. P. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol. 1992 May;6(9):1195–1204. doi: 10.1111/j.1365-2958.1992.tb01558.x. [DOI] [PubMed] [Google Scholar]
  24. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  25. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  28. Wattiau P., Cornelis G. R. SycE, a chaperone-like protein of Yersinia enterocolitica involved in Ohe secretion of YopE. Mol Microbiol. 1993 Apr;8(1):123–131. doi: 10.1111/j.1365-2958.1993.tb01209.x. [DOI] [PubMed] [Google Scholar]
  29. West S. E., Iglewski B. H. Codon usage in Pseudomonas aeruginosa. Nucleic Acids Res. 1988 Oct 11;16(19):9323–9335. doi: 10.1093/nar/16.19.9323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yahr T. L., Hovey A. K., Kulich S. M., Frank D. W. Transcriptional analysis of the Pseudomonas aeruginosa exoenzyme S structural gene. J Bacteriol. 1995 Mar;177(5):1169–1178. doi: 10.1128/jb.177.5.1169-1178.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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